A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines.

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Protective potential of sulfated polysaccharides from tropical seaweeds against alkylating- and oxidizing-induced genotoxicity.

Sulfated polysaccharides (SPs) from seaweeds are potential bioactive natural compounds, but their DNA protective activity is poorly explored. This article aimed to evaluate the genotoxic/antigenotoxic potentials of a sulfated heterofucan from brown seaweed Spatoglossum schröederi (Fucan A - FA) and a sulfated galactan from green seaweed Codium isthomocladum (3G4S) using in vitro Comet assay (alkaline and oxidative versions) with HepG2 cells. The antioxidant activity of these SPs was evaluated by total antioxidant capacity, radical scavenging, metal chelating, and antioxidant enzyme activity assays. Both SPs were not genotoxic. FA and 3G4S displayed strong antigenotoxic activity against oxidizing chemical (H2O2) but not against alkylating chemical (MMS). The DNA damage reduction after a pre-treatment of 72 h with these SPs was 81.42% to FA and 81.38% to 3G4S. In simultaneous exposure to FA or 3G4S with H2O2, HepG2 cells presented 48.04% and 55.41% of DNA damage reduction compared with the control, respectively. The antigenotoxicity of these SPs relates to direct antioxidant activity by blockage of the initiation step of the oxidative chain reaction. Therefore, we conclude that FA and 3G4S could be explored as functional natural compounds with antigenotoxic activity due to their great protection against oxidative DNA damage.

Development of 3D cultures of zebrafish liver and embryo cell lines: a comparison of different spheroid formation methods.

Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3-5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia.

Slow delivery of biocide from nanostructured, microscaled, particles reduces its phytoxicity: A model investigation.

Nano-engineered delivery systems have emerged as possible solutions for more efficient pest management in agriculture. Likewise for nanostructured drug delivery systems (DDS) in medicine, the use of biocide delivery systems (BDS) brought concerns on their toxicology on non-targeted organisms. Plants, for instance, are the foundation of the ecosystem, acting as primary actor in the food chain and is associated with the whole biodiversity, being strictly related to human health. This is a very important consideration to fully understand the benefits of using delivery systems for crop protection and production. Herein, a biocide delivery system was prepared by loading nanostructured, microscaled, biogenic silica particles with thymol, a known phytotoxicant. The resulting system contains 120 mg of thymol per gram of silica and displays slow release features. The Allium cepa bioassay was chosen to demonstrate how the toxicity and cellular damages induced by thymol can be significantly reduced through a slow, controlled, release strategy. The lower mobility of the reference particles associated with slow-delivery features reduced the toxicity and cellular damages caused by thymol in the plant genetic model.